Learning from Yeast about Mitochondrial Carriers

Mitochondria are organelles that play an important role in both energetic and synthetic metabolism of eukaryotic cells. The flow of metabolites between the cytosol and mitochondrial matrix is controlled by a set of highly selective carrier proteins localised in the inner mitochondrial membrane. As defects in the transport of these molecules may affect cell metabolism, mutations in genes encoding for mitochondrial carriers are involved in numerous human diseases. Yeast Saccharomyces cerevisiae is a traditional model organism with unprecedented impact on our understanding of many fundamental processes in eukaryotic cells. As such, the yeast is also exceptionally well suited for investigation of mitochondrial carriers. This article reviews the advantages of using yeast to study mitochondrial carriers with the focus on addressing the involvement of these carriers in human diseases.

Mitochondrial Carriers and Substrates Transport Network: A Lesson from Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is one of the most widely used model organisms for investigating various aspects of basic cellular functions that are conserved in human cells. This organism, as well as human cells, can modulate its metabolism in response to specific growth conditions, different environmental changes, and nutrient depletion. This adaptation results in a metabolic reprogramming of specific metabolic pathways. Mitochondrial carriers play a fundamental role in cellular metabolism, connecting mitochondrial with cytosolic reactions. By transporting substrates across the inner membrane of mitochondria, they contribute to many processes that are central to cellular function. The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, most of which have been functionally characterized. The aim of this review is to describe the role of the so far identified yeast mitochondrial carriers in cell metabolism, attempting to show the functional connections between substrates transport and specific metabolic pathways, such as oxidative phosphorylation, lipid metabolism, gluconeogenesis, and amino acids synthesis. Analysis of the literature reveals that these proteins transport substrates involved in the same metabolic pathway with a high degree of flexibility and coordination. The understanding of the role of mitochondrial carriers in yeast biology and metabolism could be useful for clarifying unexplored aspects related to the mitochondrial carrier network. Such knowledge will hopefully help in obtaining more insight into the molecular basis of human diseases.

Structural Mechanism of Transport of Mitochondrial Carriers

Members of the mitochondrial carrier family [solute carrier family 25 (SLC25)] transport nucleotides, amino acids, carboxylic acids, fatty acids, inorganic ions, and vitamins across the mitochondrial inner membrane. They are important for many cellular processes, such as oxidative phosphorylation of lipids and sugars, amino acid metabolism, macromolecular synthesis, ion homeostasis, cellular regulation, and differentiation. Here, we describe the functional elements of the transport mechanism of mitochondrial carriers, consisting of one central substrate-binding site and two gates with salt-bridge networks on either side of the carrier. Binding of the substrate during import causes three gate elements to rotate inward, forming the cytoplasmic network and closing access to the substrate-binding site from the intermembrane space. Simultaneously, three core elements rock outward, disrupting the matrix network and opening the substrate-binding site to the matrix side of the membrane. During export, substrate binding triggers conformational changes involving the same elements but operating in reverse. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Sequence Features of Mitochondrial Transporter Protein Families

Mitochondrial carriers facilitate the transfer of small molecules across the inner mitochondrial membrane (IMM) to support mitochondrial function and core cellular processes. In addition to the classical SLC25 (solute carrier family 25) mitochondrial carriers, the past decade has led to the discovery of additional protein families with numerous members that exhibit IMM localization and transporter-like properties. These include mitochondrial pyruvate carriers, sideroflexins, and mitochondrial cation/H+ exchangers. These transport proteins were linked to vital physiological functions and disease. Their structures and transport mechanisms are, however, still largely unknown and understudied. Protein sequence analysis per se can often pinpoint hotspots that are of functional or structural importance. In this review, we summarize current knowledge about the sequence features of mitochondrial transporters with a special focus on the newly included SLC54, SLC55 and SLC56 families of the SLC solute carrier superfamily. Taking a step further, we combine sequence conservation analysis with transmembrane segment and secondary structure prediction methods to extract residue positions and sequence motifs that likely play a role in substrate binding, binding site gating or structural stability. We hope that our review will help guide future experimental efforts by the scientific community to unravel the transport mechanisms and structures of these novel mitochondrial carriers.

Mitochondrial Carriers Regulating Insulin Secretion Profiled in Human Islets upon Metabolic Stress

Chronic exposure of β-cells to nutrient-rich metabolic stress impairs mitochondrial metabolism and its coupling to insulin secretion. We exposed isolated human islets to different metabolic stresses for 3 days: 0.4 mM oleate or 0.4 mM palmitate at physiological 5.5 mM glucose (lipotoxicity), high 25 mM glucose (glucotoxicity), and high 25 mM glucose combined with 0.4 mM oleate and/or palmitate (glucolipotoxicity). Then, we profiled the mitochondrial carriers and associated genes with RNA-Seq. Diabetogenic conditions, and in particular glucotoxicity, increased expression of several mitochondrial solute carriers in human islets, such as the malate carrier DIC, the α-ketoglutarate-malate exchanger OGC, and the glutamate carrier GC1. Glucotoxicity also induced a general upregulation of the electron transport chain machinery, while palmitate largely counteracted this effect. Expression of different components of the TOM/TIM mitochondrial protein import system was increased by glucotoxicity, whereas glucolipotoxicity strongly upregulated its receptor subunit TOM70. Expression of the mitochondrial calcium uniporter MCU was essentially preserved by metabolic stresses. However, glucotoxicity altered expression of regulatory elements of calcium influx as well as the Na+/Ca2+ exchanger NCLX, which mediates calcium efflux. Overall, the expression profile of mitochondrial carriers and associated genes was modified by the different metabolic stresses exhibiting nutrient-specific signatures.

Sequence Features of Mitochondrial Transporter Protein Families

Mitochondrial carriers facilitate the transfer of small molecules across the inner mitochondrial membrane (IMM) to support mitochondrial function and core cellular processes. In addition to the classical mitochondrial carrier family SLC25, the past decade led to the discovery of additional protein families that exhibit IMM localization and transporter-like properties. These include mitochondrial pyruvate carriers, sideroflexins and mitochondrial cation/H+ exchangers that have been linked to vital physiological functions and disease. Their structures and transport mechanisms are still largely unknown and understudied. Protein sequence analysis per se can often pinpoint hotspots that are of functional or structural importance. In this review, we summarize current knowledge about sequence features of mitochondrial transporters with a special focus on the newly included SLC54, SLC55 and SLC56 families of the SLC solute carrier superfamily. Taking a step further, we combined sequence conservation analysis with transmembrane segment and secondary structure prediction methods to extract residue positions and sequence motifs that likely play a role in substrate binding, binding site gating or structural stability. We hope that our review will help guide future experimental efforts by the scientific community to unravel the transport mechanism and structure of these novel mitochondrial carriers.

Drosophila melanogaster Mitochondrial Carriers: Similarities and Differences with the Human Carriers

Mitochondrial carriers are a family of structurally related proteins responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. The in silico analysis of the Drosophila melanogaster genome has highlighted the presence of 48 genes encoding putative mitochondrial carriers, but only 20 have been functionally characterized. Despite most Drosophila mitochondrial carrier genes having human homologs and sharing with them 50% or higher sequence identity, D. melanogaster genes display peculiar differences from their human counterparts: (1) in the fruit fly, many genes encode more transcript isoforms or are duplicated, resulting in the presence of numerous subfamilies in the genome; (2) the expression of the energy-producing genes in D. melanogaster is coordinated from a motif known as Nuclear Respiratory Gene (NRG), a palindromic 8-bp sequence; (3) fruit-fly duplicated genes encoding mitochondrial carriers show a testis-biased expression pattern, probably in order to keep a duplicate copy in the genome. Here, we review the main features, biological activities and role in the metabolism of the D. melanogaster mitochondrial carriers characterized to date, highlighting similarities and differences with their human counterparts. Such knowledge is very important for obtaining an integrated view of mitochondrial function in D. melanogaster metabolism.

Metabolic Roles of Plant Mitochondrial Carriers

Mitochondrial carriers (MC) are a large family (MCF) of inner membrane transporters displaying diverse, yet often redundant, substrate specificities, as well as differing spatio-temporal patterns of expression; there are even increasing examples of non-mitochondrial subcellular localization. The number of these six trans-membrane domain proteins in sequenced plant genomes ranges from 39 to 141, rendering the size of plant families larger than that found in Saccharomyces cerevisiae and comparable with Homo sapiens. Indeed, comparison of plant MCs with those from these better characterized species has been highly informative. Here, we review the most recent comprehensive studies of plant MCFs, incorporating the torrent of genomic data emanating from next-generation sequencing techniques. As such we present a more current prediction of the substrate specificities of these carriers as well as review the continuing quest to biochemically characterize this feature of the carriers. Taken together, these data provide an important resource to guide direct genetic studies aimed at addressing the relevance of these vital carrier proteins.